Methods for treating heart disease in a subject with Friedreich&#39;s ataxia by an aromatic-cationic peptide

ABSTRACT

The disclosure provides methods of preventing or treating Friedreich&#39;s ataxia in a mammalian subject, reducing risk factors, signs and/or symptoms associated with Friedreich&#39;s ataxia, and/or reducing the likelihood or severity of Friedreich&#39;s ataxia. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to e.g., reduce oxidative stress, increase mitochondrial metabolism, or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.16/425,892, filed May 29, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/697,648, filed Sep. 7, 2017, which is acontinuation of U.S. patent application Ser. No. 14/908,053, filed Jan.27, 2016, which is the U.S. 371 National Stage Application ofPCT/2014/049633, filed Aug. 4, 2014, which claims the benefit of andpriority to U.S. Application No. 61/861,806, filed Aug. 2, 2013, theentire contents of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present technology relates generally to compositions and methods forameliorating or treating Friedreich's ataxia and/or reducing theseverity of Friedreich's ataxia. In particular, the present technologyrelates to administering an effective amount of an aromatic-cationicpeptide to a subject suffering from Friedreich's ataxia.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the compositions and methods disclosed herein.

Friedreich's ataxia is an inherited autosomal recessive disease thatcauses progressive damage to the nervous system. The ataxia results fromthe degeneration of nerve tissue in the spinal cord, in particular,sensory neurons essential for directing muscle movement of the arms andlegs. The spinal cord becomes thinner and nerve cells lose some of theirmyelin sheath.

Friedreich's ataxia occurs when the FXN gene contains amplified intronicGAA repeats. The mutant FXN gene contains expanded GAA triplet repeatsin the first intron; in a few pedigrees, point mutations have also beendetected. Since the defect is located in an intron, which is removedfrom the mRNA transcript between transcription and translation, themutated FXN gene does not result in the production of abnormal proteins.Instead, the mutation causes gene silencing, i.e., the mutationdecreases the transcription of the gene, through induction of aheterochromatin structure in a manner similar to position-effectvariegation.

The FXN gene encodes the protein frataxin. GAA repeat expansion causesfrataxin levels to be reduced. Frataxin is an iron binding proteinresponsible for forming iron-sulphur clusters. One result of frataxindeficiency is mitochondrial iron overload.

SUMMARY

In one aspect, the present disclosure provides methods for treating orpreventing Friedreich's ataxia, and/or treating or preventing the signsor symptoms of reduced levels of frataxin or frataxin activity in asubject in need thereof by administering to the subject atherapeutically effective amount of an aromatic-cationic peptide such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.

In some embodiments, the subject displays reduced levels of frataxincompared to a normal control subject.

In some embodiments, the peptide is administered daily for 6 weeks ormore. In some embodiments, the peptide is administered daily for 12weeks or more.

In some embodiments, the subject has been diagnosed as havingFriedreich's ataxia.

In some embodiments, the Friedreich's ataxia includes one or more ofmuscle weakness, loss of coordination, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders.

In some embodiments, the subject is human.

In some embodiments, the peptide is administered orally, topically,systemically, intravenously, subcutaneously, intraperitoneally, orintramuscularly

In some embodiments, the method also includes separately, sequentiallyor simultaneously administering to the subject one or more agentsselected from the group consisting of ACE inhibitors, digoxin,enalapril, or lisinopril, diuretics, beta blockers, idebenone,deferiprone, and insulin. In some embodiments, there is a synergisticeffect between the peptide and the additional agent in this regard.

In some embodiments, the pharmaceutically acceptable salt comprisesacetate or trifluoroacetate salt.

In one aspect, the present technology provides a method for reducingmitochondrial iron in a mammalian subject having or suspected of havingFriedreich's ataxia, the method comprising: administering to the subjecta therapeutically effective amount of the peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof. In some embodiments, the mammalian subject has decreasedexpression of frataxin compared to a normal control subject. In someembodiments, the subject is human.

In some embodiments, the peptide is administered daily for 6 weeks ormore. In some embodiments, the peptide is administered daily for 12weeks or more.

In some embodiments, the Friedreich's ataxia comprises one or more ofmuscle weakness, loss of coordination, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders.

In some embodiments, the peptide is administered orally, topically,systemically, intravenously, subcutaneously, intraperitoneally, orintramuscularly

In some embodiments, the method includes administering separately,sequentially or simultaneously to the subject one or more therapeuticagents selected from the group consisting of ACE inhibitors, digoxin,enalapril, or lisinopril, diuretics, beta blockers, idebenone,deferiprone, and insulin.

In some embodiments, the pharmaceutically acceptable salt comprisesacetate or trifluoroacetate salt.

In some embodiments, the combination of peptide and an additionaltherapeutic agent has a synergistic effect in the reduction ofmitochondrial iron and/or prevention or treatment of Friedreich'sataxia.

In one aspect, the present technology provides for methods for reducingthe risk, signs or symptoms of Friedreich's ataxia in a mammaliansubject having decreased expression of frataxin compared to a normalcontrol subject. In some embodiments, the method includes administeringto the subject a therapeutically effective amount of the peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.

In one aspect, the present technology provides for methods ofstabilizing mitochondrial metabolism in a mammalian subject having orsuspected of having Friedreich's ataxia and/or having lower than controlor normal levels of frataxin. In some embodiments, the method includesadministering to the subject a therapeutically effective amount of thepeptide D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptablesalt thereof.

In one aspect, the disclosure provides a method of treating orpreventing Friedreich's ataxia in a mammalian subject, comprisingadministering to said mammalian subject a therapeutically effectiveamount of an aromatic-cationic peptide. In some embodiments, thearomatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1. In particular embodiments, the mammalian subject isa human.

In one embodiment, 2p_(m) is the largest number that is less than orequal to r+1, and may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In one embodiment, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In a particularembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m=1-3;

(iv)

(v)

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

where m=1-3;

(iv)

(v)

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered orally, topically,intranasally, intravenously, subcutaneously, or transdermally (e.g., byiontophoresis).

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent technology. The definitions of certain terms as used in thisspecification are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this technology belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, butthose functions in a manner similar to a naturally occurring amino acid.Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g. an amount that reduces, ameliorates or delays the onset of thephysiological symptoms of Friedreich's ataxia. In the context oftherapeutic or prophylactic applications, in some embodiments, theamount of a composition administered to the subject will depend on thetype and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. In some embodiments, it will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds. In the methods described herein,aromatic-cationic peptides, such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt, may be administered to a subject having one ormore signs, symptoms, or risk factors of Friedreich's ataxia, such as,e.g., muscle weakness, especially in the arms and legs, loss ofcoordination, motor control impairment, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders. For example, a “therapeutically effective amount” of thearomatic-cationic peptides includes levels at which the presence,frequency, or severity of one or more signs, symptoms, or risk factorsof Friedreich's ataxia are reduced or eliminated. In some embodiments, atherapeutically effective amount reduces or ameliorates thephysiological effects of Friedreich's ataxia, and/or the risk factors ofFriedreich's ataxia, and/or delays the progression or onset ofFriedreich's ataxia.

As used herein, “isolated” or “purified” polypeptide or peptide refersto a polypeptide or peptide that is substantially free of cellularmaterial or other contaminating polypeptides from the cell or tissuesource from which the agent is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized. Forexample, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this definition.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to therapeutic treatment, wherein the object is to reduce,alleviate or slow down (lessen) the targeted pathologic condition ordisorder. By way of example, but not by way of limitation, a subject issuccessfully “treated” for Friedreich's ataxia if, after receiving atherapeutic amount of the aromatic-cationic peptides, such asD-Arg-2′6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, according to themethods described herein, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms ofFriedreich's ataxia, such as but not limited to, e.g., muscle weakness,especially in the arms and legs, loss of coordination, motor controlimpairment, vision impairment, hearing impairment, slurred speech,curvature of the spine, diabetes, and heart disorders. It is also to beappreciated that the various modes of treatment of medical conditions asdescribed are intended to mean “substantial,” which includes total butalso less than total treatment, and wherein some biologically ormedically relevant result is achieved. Treating Friedreich's ataxia, asused herein, also refers to treating the signs and symptoms related toreduced frataxin activity or frataxin expression levels characteristicof Friedreich's ataxia.

As used herein, “prevention” or “preventing” of a disease or condition,e.g., Friedreich's ataxia refers to results that, in a statisticalsample, exhibit a reduction in the occurrence of the disorder orcondition in the treated sample relative to an untreated control sample,or exhibit a delay in the onset of one or more symptoms of the disorderor condition relative to the untreated control sample. As used herein,preventing Friedreich's ataxia includes preventing or delaying theinitiation of, preventing, delaying, or slowing the progression oradvancement of Friedreich's ataxia. As used herein, prevention ofFriedreich's ataxia also includes preventing a recurrence of one or moresigns or symptoms of Friedreich's ataxia.

Aromatic-Cationic Peptides

The present technology relates to methods and compositions forpreventing or treating Friedreich's ataxia in a subject in need thereof.In some embodiments, the methods and compositions prevent one or moresigns or symptoms of Friedreich's ataxia in a subject. In someembodiments, the methods and compositions increase the level of frataxinexpression in a subject. In some embodiments, the methods andcompositions reduce the likelihood that a subject with risk factors forFriedreich's ataxia will develop one or more signs or symptoms ofFriedreich's ataxia, or will delay the onset of Friedreich's ataxia. Insome embodiments, the methods and compositions include anaromatic-cationic peptide such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt.

It is known in the art that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, possess anti-oxidantproperties, including the capacity to reduce the rate of lipidoxidation, peroxidation, mitochondrial H₂O₂ production, andintracellular reactive oxygen species (ROS) production. It is furtherknown in the art that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, localize to themitochondria, and have the capacity to inhibit caspase activation andapoptosis. It has also been shown that aromatic-cationic peptides, suchas D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, restore mitochondria membrane potential.These and other properties of aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, are demonstrated inU.S. application Ser. No. 11/040,242 (U.S. Pat. No. 7,550,439) and Ser.No. 10/771,232 (U.S. Pat. No. 7,576,061). Accordingly, aromatic-cationicpeptides of the present technology, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂,are useful in the prevention and treatment of diseases and conditionscaused by, resulting from, or otherwise associated with such cellularevents, such as Friedreich's ataxia.

The aromatic-cationic peptides are water-soluble and highly polar.Despite these properties, the peptides can readily penetrate cellmembranes. The aromatic-cationic peptides typically include a minimum ofthree amino acids or a minimum of four amino acids, covalently joined bypeptide bonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, about nine, or about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the α positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. Optimally, the peptide has no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta-, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are suitably resistant orinsensitive to common proteases. Examples of non-naturally occurringamino acids that are resistant or insensitive to proteases include thedextrorotatory (D-) form of any of the above-mentioned naturallyoccurring L-amino acids, as well as L- and/or D-non-naturally occurringamino acids. The D-amino acids do not normally occur in proteins,although they are found in certain peptide antibiotics that aresynthesized by means other than the normal ribosomal protein syntheticmachinery of the cell. As used herein, the D-amino acids are consideredto be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, less than four, less than three, or less than two contiguousL-amino acids recognized by common proteases, irrespective of whetherthe amino acids are naturally or non-naturally occurring. Optimally, thepeptide has only D-amino acids, and no L-amino acids. If the peptidecontains protease sensitive sequences of amino acids, at least one ofthe amino acids is, in some embodiments, a non-naturally-occurringD-amino acid, thereby conferring protease resistance. An example of aprotease sensitive sequence includes two or more contiguous basic aminoacids that are readily cleaved by common proteases, such asendopeptidases and trypsin. Examples of basic amino acids includearginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below is all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 2 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 3 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges or a minimum of three net positive charges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 5 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

TABLE 6 EXEMPLARY PEPTIDES 2′,6′-Dmp-D-Arg-2′,6′-Dmt-Lys-NH₂2′,6′-Dmp-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Arg-Phe-Orn-NH₂2′,6′-Dmt-D-Arg-Phe-Ahp(2-aminoheptanoicacid)-NH₂2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ 2′,6′-Dmt-D-Cit-PheLys-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheArg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D- Arg-GlyAsp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-Arg-2′,6′-Dmt-Lys-Phe-NH₂D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂ D-His-Glu-Lys-Tyr-D-Phe-ArgD-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-Asp-D-His-D-Lys-Arg-Trp-NH₂D-Tyr-Trp-Lys-NH₂Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D- Met-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp. Gly-D-Phe-Lys-His-D-Arg-Tyr-NH₂His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂ Lys-D-Arg-Tyr-NH₂ Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂ Met-Tyr-D-Arg-Phe-Arg-NH₂Met-Tyr-D-Lys-Phe-Arg Phe-Arg-D-His-Asp Phe-D-Arg-2′,6′-Dmt-Lys-NH₂Phe-D-Arg-His Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His Phe-D-Arg-Phe-Lys-NH₂Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrThr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysTyr-D-Arg-Phe-Lys-Glu-NH₂ Tyr-D-Arg-Phe-Lys-NH₂Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe Tyr-His-D-Gly-MetVal-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂

In one embodiment, the peptides have mu-opioid receptor agonist activity(i.e., they activate the mu-opioid receptor). Peptides, which havemu-opioid receptor agonist activity, are typically those peptides thathave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Suitable derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′,6′-Dmt);3′,5′-dimethyltyrosine (3′,5′-Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂. Tyr-D-Arg-Phe-Lys-NH₂has a net positive charge of three, contributed by the amino acidstyrosine, arginine, and lysine and has two aromatic groups contributedby the amino acids phenylalanine and tyrosine. The tyrosine ofTyr-D-Arg-Phe-Lys-NH₂ can be a modified derivative of tyrosine such asin 2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂. 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ has a molecularweight of 640 and carries a net three positive charge at physiologicalpH. 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ readily penetrates the plasma membraneof several mammalian cell types in an energy-independent manner (Zhao,et al., J. Pharmacol Exp Ther., 304:425-432, 2003).

Alternatively, in other instances, the aromatic-cationic peptide doesnot have mu-opioid receptor agonist activity. For example, duringlong-term treatment, such as in a chronic disease state or condition,the use of an aromatic-cationic peptide that activates the mu-opioidreceptor may be contraindicated. In these instances, the potentiallyadverse or addictive effects of the aromatic-cationic peptide maypreclude the use of an aromatic-cationic peptide that activates themu-opioid receptor in the treatment regimen of a human patient or othermammal. Potential adverse effects may include sedation, constipation andrespiratory depression. In such instances an aromatic-cationic peptidethat does not activate the mu-opioid receptor may be an appropriatetreatment. Peptides that do not have mu-opioid receptor agonist activitygenerally do not have a tyrosine residue or a derivative of tyrosine atthe N-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N, 2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂.Alternatively, the N-terminal phenylalanine can be a derivative ofphenylalanine such as 2′,6′-dimethylphenylalanine (2′,6′-Dmp).Tyr-D-Arg-Phe-Lys-NH₂ containing 2′,6′-dimethylphenylalanine at aminoacid position 1 has the formula 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In oneembodiment, the amino acid sequence of 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ isrearranged such that Dint is not at the N-terminus. An example of suchan aromatic-cationic peptide that does not have mu-opioid receptoragonist activity has the formula D-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

Suitable substitution variants of the peptides listed herein includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group are referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group are generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 7.

TABLE 7 Peptide Analogs with Mu-Opioid Activity Amino Amino Amino AminoAcid Acid Acid Acid Position Position Position Position C-Terminal 1 2 34 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ Tyr D-Arg PheDab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-ArgPhe Lys-NH(CH₂)₂-NH-dns NH₂ 2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂-NH-atn NH₂2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit PheAhp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′DmtD-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Ahp(2-aminoheptanoic NH₂ acid) Bio-D-Arg Phe Lys NH₂ 2′6′Dmt 3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg PheOrn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-ArgTyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg TyrDap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′DmtD-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt LysNH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′DmtD-Arg 2′6′Dmt Dap NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt DabNH₂ Tyr D-Lys Phe Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂Tyr D-Lys Phe Orn NH₂ 2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe DapNH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-LysPhe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′DmtD-Lys Phe Arg NH₂ Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-LysTyr Dab NH₂ Tyr D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′DmtD-Lys Tyr Orn NH₂ 2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂2′6′Dmt D-Lys 2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′DmtD-Lys 2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg PhednsDap NH₂ 2′6′Dmt D-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂3′5′Dmt D-Lys 3′5′Dmt Orn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′DmtD-Lys 3′5′Dmt Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ TyrD-Dab Phe Arg NH₂ Tyr D-Dap Phe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂2′6′Dmt D-Lys Phe Arg NH₂ 2′6′Dmt D-Orn Phe Arg NH₂ 2′6′Dmt D-Dab PheArg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂ 3′5′Dmt D-Arg Phe Arg NH₂ 3′5′DmtD-Lys Phe Arg NH₂ 3′5′Dmt D-Orn Phe Arg NH₂ Tyr D-Lys Tyr Arg NH₂ TyrD-Orn Tyr Arg NH₂ Tyr D-Dab Tyr Arg NH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′DmtD-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂ 3′5′Dmt D-Dap 3′5′Dmt ArgNH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Lys 3′5′Dmt Arg NH₂ 3′5′DmtD-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe Lys NH₂ Mmt D-Arg Phe Orn NH₂ MmtD-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂ Tmt D-Arg Phe Lys NH₂ Tmt D-ArgPhe Orn NH₂ Tmt D-Arg Phe Dab NH₂ Tmt D-Arg Phe Dap NH₂ Hmt D-Arg PheLys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-Arg Phe Dab NH₂ Hmt D-Arg Phe DapNH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys Phe Orn NH₂ Mmt D-Lys Phe Dab NH₂Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe Arg NH₂ Tmt D-Lys Phe Lys NH₂ TmtD-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂ Tmt D-Lys Phe Dap NH₂ Tmt D-LysPhe Arg NH₂ Hmt D-Lys Phe Lys NH₂ Hmt D-Lys Phe Orn NH₂ Hmt D-Lys PheDab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-Lys Phe Arg NH₂ Mmt D-Lys Phe ArgNH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap Phe Arg NH₂Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe Arg NH₂ TmtD-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂ Hmt D-LysPhe Arg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ Hmt D-Dap PheArg NH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric Dap =diaminopropionic acid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt= N, 2′,6′-trimethyltyrosine Hmt = 2′-hydroxy,6′-methyltyrosine dnsDap =β-dansyl-L-α,β-diaminopropionic acid atnDap =β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 8.

TABLE 8 Peptide Analogs Lacking Mu-Opioid Activity Amino Amino AminoAmino Acid Acid Acid Acid Position Position Position Position C-Terminal1 2 3 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂ D-ArgPhe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-Arg LysPhe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-Arg PheLys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-Arg LysNH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-Arg NH₂Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂ LysD-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂ D-ArgDmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂ TrpD-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-Arg TrpLys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg Trp DmtLys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg Phe LysNH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc., New York (1997).

Friedreich's Ataxia

Friedreich's ataxia is an inherited autosomal recessive disease thatcauses progressive damage to the nervous system. The ataxia results fromthe degeneration of nerve tissue in the spinal cord, in particular,sensory neurons essential for directing muscle movement of the arms andlegs. The spinal cord becomes thinner and nerve cells lose some of theirmyelin sheath.

Symptoms typically begin between the ages of 5 and 15 years, althoughthey sometimes appear in adulthood. The first symptom to appear isusually gait ataxia, or difficulty walking. The ataxia gradually worsensand slowly spreads to the arms and the trunk. There is often loss ofsensation in the extremities, which may spread to other parts of thebody. Other features include loss of tendon reflexes, especially in theknees and ankles. Most people with Friedreich's ataxia developscoliosis, which often requires surgical intervention for treatment.Dysarthria (slowness and slurring of speech) develops and can getprogressively worse. Many individuals with later stages of Friedreich'sataxia develop hearing and vision loss.

Heart disease often accompanies Friedreich's ataxia, such ashypertrophic cardiomyopathy (enlargement of the heart), myocardialfibrosis (formation of fiber-like material in the muscles of the heart),and cardiac failure. Heart rhythm abnormalities such as tachycardia(fast heart rate) and heart block (impaired conduction of cardiacimpulses within the heart) are also common. Other symptoms that mayoccur include chest pain, shortness of breath, and heart palpitations.

About 20 percent of people with Friedreich's ataxia develop carbohydrateintolerance and 10 percent develop diabetes. Most individuals withFriedreich's ataxia tire very easily and find that they require morerest and take a longer time to recover from common illnesses such ascolds and flu.

The rate of progression varies from person to person. Generally, within10 to 20 years after the appearance of the first symptoms, the person isconfined to a wheelchair, and in later stages of the disease individualsmay become completely incapacitated. Friedreich's ataxia can shortenlife expectancy, and heart disease is the most common cause of death.

Friedreich's ataxia occurs when a mutated FXN gene contains amplifiedintronic GAA repeats. The mutant FXN gene contains expanded GAA tripletrepeats in the first intron; in a few pedigrees, point mutations havebeen detected. Since the defect is located in an intron, which isremoved from the mRNA transcript between transcription and translation,the mutated FXN gene does not result in the production of abnormalproteins. Instead, the mutation causes gene silencing, i.e., themutation decreases the transcription of the gene, through induction of aheterochromatin structure in a manner similar to position-effectvariegation. The GAA repeat expansion in FXN and subsequent genesilencing results in the reduction of frataxin levels.

The FXN gene encodes the protein frataxin. Frataxin is a highlyconserved iron binding protein. Human frataxin is synthesized as a 210amino acid precursor that is imported to the mitochondria via themitochondrial targeting signal contained in the N-terminus. The frataxinprecursor is subsequently cleaved to a mature 14 kDa protein (residues81-210).

Frataxin binds both Fe²⁺ and Fe³⁺ ions in an electrostatic manner andfunctions as an iron chaperone during Fe—S cluster assembly. Frataxindirectly binds to the central Fe—S cluster assembly complex, which iscomposed of Nfs1 enzyme and Isu scaffold protein. Nfs1 is a cysteinedesulfurase used in the synthesis of sulfur bioorganic derivatives andIsu is the transient scaffold protein on which the Fe—S clusterassembles. Frataxin increases the efficiency of Fe—S cluster formation,which is required to activate the mitochondrial Kreb cycle enzymeaconitase. Frataxin also plays a role in mitochondrial iron storage andheme biosynthesis by incorporating mitochondrial iron intoprotoporphyrin (PIX).

Loss of frataxin function results in the disruption of iron-sulfurcluster biosynthesis, mitochondrial iron overload, oxidative stress,impaired aerobic electron transport chain respiration and cell death inthe brain, spinal cord and heart. Studies have also shown that frataxinprotects dopaminergic neuronal cells against MPTP-induced toxicity in amouse model of Parkinson's disease.

Mitochondrial iron overload leads to impaired intra-mitochondrialmetabolism and a defective mitochondrial respiratory chain. A defectivemitochondrial respiratory chain leads to increased free radicalgeneration and oxidative damage, which may be considered as mechanismsthat compromise cell viability. Recent evidence suggests that frataxinmight detoxify reactive oxygen species (ROS) via activation ofglutathione peroxidase and elevation of thiols. (See e.g., Calabrese etal., Journal of the Neurological Sciences, 233(1): 145-162 (June 2005)).

In some embodiments, treatment with an aromatic-cationic peptide, suchas D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, stabilizes themitochondrial metabolism in a tissue or an organ in mammalian subjectsthat have suffered or are at risk of suffering Friedreich's ataxia. Byway of example, but not by way of limitation, in some embodiments,mitochondrial metabolism is increased in the spinal cord of a treatedsubject.

In some embodiments, treatment with an aromatic-cationic peptide, suchas D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, reduces free radicalgeneration, oxidative stress, or both in a tissue or an organ inmammalian subjects that have suffered or are at risk of sufferingFriedreich's ataxia. By way of example, but not by way of limitation, insome embodiments, free radical generation, oxidative damage, or both areincreased in the spinal cord of a treated subject.

In some embodiments, treatment with an aromatic-cationic peptide, suchas D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, reduces build-up ofiron in the mitochondria in a tissue or an organ in mammalian subjectsthat have suffered or are at risk of suffering Friedreich's ataxia. Byway of example, but not by way of limitation, in some embodiments, ironin the mitochondria decreases in the spinal cord of a treated subject.

Therapeutic Methods

The following discussion is presented by way of example only, and is notintended to be limiting.

One aspect of the present technology includes methods of treatingreduced frataxin expression in a subject diagnosed as having, suspectedas having, or at risk of having reduced frataxin expression levels. Oneaspect of the present technology includes methods of treatingFriedreich's ataxia in a subject diagnosed as having, suspected ashaving, or at risk of having Friedreich's ataxia. In therapeuticapplications, compositions or medicaments comprising anaromatic-cationic peptide such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt, are administered to a subject suspected of, oralready suffering from such a disease, such as, e.g., decreased frataxinexpression levels or Friedreich's ataxia, in an amount sufficient toreduce the severity at least partially arrest or delay the onset of oneor more of the symptoms of the disease, including its complications andintermediate pathological phenotypes in development of the disease.

Subjects suffering from decreased frataxin expression levels orFriedreich's ataxia can be identified by any or a combination ofdiagnostic or prognostic assays known in the art. For example, typicalsymptoms of Friedreich's ataxia include symptoms such as, e.g., muscleweakness, especially in the arms and legs, loss of coordination, motorcontrol impairment, vision impairment, hearing impairment, slurredspeech, curvature of the spine, diabetes, and heart disorders. In someembodiments, the subject may exhibit reduced levels of frataxinexpression compared to a normal subject, which are measureable usingtechniques known in the art. In some embodiments, the subject mayexhibit one or more mutations in the FXN gene associated withFriedreich's ataxia, which are detectable using techniques known in theart.

Prophylactic Methods

In one aspect, the present technology provides a method for preventingor delaying the onset of Friedreich's ataxia or symptoms of Friedreich'sataxia in a subject at risk of having reduced levels of frataxinexpression compared to a normal subject. In some embodiments, thesubject may exhibit one or more mutations in the FXN gene associatedwith Friedreich's ataxia, which are detectable using techniques known inthe art. Subjects at risk for reduced frataxin expression levels orFriedreich's ataxia can be identified by, e.g., any or a combination ofdiagnostic or prognostic assays known in the art. In prophylacticapplications, pharmaceutical compositions or medicaments ofaromatic-cationic peptides, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt, are administered to a subject susceptible to, orotherwise at risk of a disease or condition such as e.g., Friedreich'sataxia, in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease. Administration of a prophylactic aromatic-cationic canoccur prior to the manifestation of symptoms characteristic of thedisease or disorder, such that the disease or disorder is prevented or,alternatively, delayed in its progression.

Subjects or at risk for reduced frataxin expression levels orFriedreich's ataxia may exhibit one or more of the followingnon-limiting risk factors: cardiomyopathy, skeletal muscleabnormalities, neutropenia, slow development, weak muscle tone,increased levels of organic acids in the urine and blood, and/orfrequent bacterial infections, such as pneumonia.

For therapeutic and/or prophylactic applications, a compositioncomprising an aromatic-cationic peptide, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, is administered tothe subject. In some embodiments, the peptide composition isadministered one, two, three, four, or five times per day. In someembodiments, the peptide composition is administered more than fivetimes per day. Additionally or alternatively, in some embodiments, thepeptide composition is administered every day, every other day, everythird day, every fourth day, every fifth day, or every sixth day. Insome embodiments, the peptide composition is administered weekly,bi-weekly, tri-weekly, or monthly. In some embodiments, the peptidecomposition is administered for a period of one, two, three, four, orfive weeks. In some embodiments, the peptide is administered for sixweeks or more. In some embodiments, the peptide is administered fortwelve weeks or more. In some embodiments, the peptide is administeredfor a period of less than one year. In some embodiments, the peptide isadministered for a period of more than one year.

For therapeutic and/or prophylactic applications, a compositioncomprising an aromatic-cationic peptide, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, may be administeredin combination with one or more additional agents. In some embodiments,there is a synergistic effect between the peptide and the one or moreadditional agents.

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment. In various embodiments, in vitro assays can be performedwith representative animal models, to determine if a givenaromatic-cationic peptide-based therapeutic exerts the desired effectincreasing frataxin expression, and preventing or treating Friedreich'sataxia. Compounds for use in therapy can be tested in suitable animalmodel systems including, but not limited to rats, mice, chicken, cows,monkeys, rabbits, and the like, prior to testing in human subjects.Similarly, for in vivo testing, any of the animal model system known inthe art can be used prior to administration to human subjects. In someembodiments, in vitro or in vivo testing is directed to the biologicalfunction of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with an aromatic-cationic peptide of the present technology, suchas D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, may be employed.Suitable methods include in vitro, ex vivo, or in vivo methods. In vivomethods typically include the administration of an aromatic-cationicpeptide, such as those described above, to a mammal, suitably a human.When used in vivo for therapy, the aromatic-cationic peptides, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, are administered tothe subject in effective amounts (i.e., amounts that have desiredtherapeutic effect). The dose and dosage regimen will depend upon thedegree of the disease in the subject, the characteristics of theparticular aromatic-cationic peptide used, e.g., its therapeutic index,the subject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods may be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide may be administeredsystemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid, which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucuronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphorsulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the salt is an acetate ortrifluoroacetate salt.

The aromatic-cationic peptides described herein, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, can be incorporatedinto pharmaceutical compositions for administration, singly or incombination, to a subject for the treatment or prevention of a disorderdescribed herein. Such compositions typically include the active agentand a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomersal, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Such methods include those described in U.S.Pat. No. 6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. s one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg, et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50.

Compounds that exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies, in some embodiments, within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to determine useful doses in humans accurately.Levels in plasma may be measured, for example, by high performanceliquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Suitably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.001-10,000 micrograms per kg body weight. In oneembodiment, aromatic-cationic peptide concentrations in a carrier rangefrom 0.2 to 2000 micrograms per delivered milliliter. An exemplarytreatment regime entails administration once per day or once a week. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and in some embodiments, until the subject showspartial or complete amelioration of symptoms of disease. Thereafter, thepatient can be administered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹² to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.001 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, and in some embodiments, by single daily or weeklyadministration, but also including continuous administration (e.g.,parenteral infusion or transdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

Combination Therapy with an Aromatic-Cationic Peptide and OtherTherapeutic Agents

In some embodiments, one or more additional therapeutic agents areadministered to a subject in combination with an aromatic-cationicpeptide, e.g., D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, suchthat a synergistic therapeutic effect is produced. A “synergistictherapeutic effect” refers to a greater-than-additive therapeuticeffect, which is produced by a combination of at least two therapeuticagents, and which exceeds that which would otherwise result fromadministration of any individual therapeutic agent alone. Therefore,lower doses of one or more of any individual therapeutic agent may beused in treating a medical disease or condition, e.g., disruptions inmitochondrial oxidative phosphorylation, resulting in increasedtherapeutic efficacy and decreased side-effects. By way of example, butnot by way of limitation, exemplary additional therapeutic agents thatcan be combined with aromatic-cationic peptides for the treatment orprevention of Friedreich's ataxia include, but are not limited to, ACEinhibitors, e.g., digoxin, enalapril, or lisinopril, diuretics,beta-blockers, idebenone, deferiprone, and insulin.

The multiple therapeutic agents may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may vary from more than zero weeks to less than fourweeks. In addition, the combination methods, compositions andformulations are not to be limited to the use of only two agents.

EXAMPLES

The present compositions and methods are further illustrated by thefollowing examples, which should not be construed as limiting in anyway.

Example 1: Aromatic-Cationic Peptides Rescue Friedreich's AtaxiaFibroblasts from Iron-Oxidant Stress

This example demonstrates the effect of the aromatic-cationic peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on fibroblasts from Friedreich's ataxia(FRDA) patients that have induced iron-oxidant stress.

Methods: In the absence of FXN, it is widely accepted that deficientcells will have an increased sensitivity to oxidative stress, which mostlikely contributes to the cascade of events leading to cytotoxicity.Iron with hydroquinone (HQ) induces oxidative stress in cells because HQforms a lipophilic chelate with iron and rapidly transfers the metalacross the normally impermeable plasma membrane. HQ or Fe alone inculture media is not toxic to FRDA fibroblasts even after an extendedexposure of 24 hours.

FRDA fibroblasts are treated with 1-10 μM D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ inculture media for 24 hours. After 24 hours, the media is changed and thecells are treated with 5 μm Fe/HQ for 5 hours. Controls include FRDAfibroblasts without D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and then treated with 5μm Fe/HQ for 5 hours and FRDA fibroblasts treated withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ for 24 hours and no addition of Fe/HQ.

Results: It is anticipated that cells that are treated only with Fc/HQwill show changes in the morphology and have loss of adherence, whichindicates that Fe/HQ is cytotoxic. It is anticipated that cells thatwere treated with D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ prior to the addition ofFe/HQ will be able to survive and show reduced evidence of cytotoxicityas demonstrated by their morphologic appearance being substantiallyidentical to, or less deformed than cells treated only withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

These results will show that aromatic-cationic peptides of the presentdisclosure, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, areuseful in the prevention and treatment of diseases and conditionsassociated with reduced frataxin expression levels. It is furtherexpected that administration of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ incombination with one or more additional therapeutic agents will havesynergistic effects in this regard. It is further anticipated that theseresults will show that aromatic-cationic peptides of the presentdisclosure, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, areuseful in methods comprising administration of the peptide to subjectshaving or susceptible to Friedreich's ataxia.

Example 2: Aromatic-Cationic Peptides Prolong Survival of FXN-KnockoutMice

This example demonstrates the effect of the aromatic-cationic peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ on survival of FXN-knockout (KO) mice.

Methods: FXN-KO mice are treated with 1-10 μMD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or PBS beginning at Day 3 of life for 60days. The mice receive the aromatic peptide and PBS by intraperitoneal(IP) injections three times per week. All mice will need to reach an ageof 10 days to be included in the study, and all mice will be weaned at28 days of age. Control animals include of littermates heterozygous forthe conditional allele and had no clinical or biochemical phenotype. Thecontrol heterozygous littermates receive equivalent volume injections ofPBS.

Results: It is anticipated that FXN-KO mice treated withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ will have increased survival as compared toFXN-KO mice treated with PBS. It is further expected that administrationof D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ in combination with one or moreadditional therapeutic agents will have synergistic effects in thisregard.

These results will show that aromatic-cationic peptides of the presentdisclosure, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, areuseful in the prevention and treatment of Friedreich's ataxia.

Example 3: Use of Aromatic-Cationic Peptides in the Treatment ofFriedreich's Ataxia

This example will demonstrate the use of aromatic-cationic peptides,such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptablesalt thereof, such as acetate or trifluoroacetate salt, in the treatmentof Friedreich's ataxia.

Methods: Friedreich's ataxia patients receive daily administrations of atherapeutically effective (e.g., 1-10 mg/kg body weight) amount ofaromatic-cationic peptide, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt. Peptides may be administered orally, topically,systemically, intravenously, subcutaneously, intraperitoneally, orintramuscularly according to methods known in the art. Subjects areevaluated weekly for the presence and/or severity of signs and symptomsassociated with Friedreich's ataxia, including, but not limited to,e.g., muscle weakness, especially in the arms and legs, loss ofcoordination, motor control impairment, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders. Treatments are maintained until such a time as symptoms ofFriedreich's ataxia are ameliorated or eliminated.

Results: It is predicted that Friedreich's ataxia subjects receivingtherapeutically effective amounts of aromatic-cationic peptide, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt will display reducedseverity of symptoms associated with Friedreich's ataxia. It is furtherexpected that administration of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ incombination with one or more additional therapeutic agents will havesynergistic effects in this regard.

These results will show that aromatic-cationic peptides, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt are useful in thetreatment of Friedreich's ataxia. Accordingly, the peptides are usefulin methods comprising administering aromatic-cationic peptides to asubject in need thereof for the treatment of Friedreich's ataxia.

Example 4: Use of Aromatic-Cationic Peptides in Combination with OtherAgents to Reduce Symptoms of Friedreich's Ataxia

This example will demonstrate the synergetic effect from the use ofaromatic-cationic peptides, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt, and another agent, e.g., idebenone, in thetreatment of Friedreich's ataxia.

Methods: Friedreich's ataxia patients are split into four groups. Group1 receives daily administrations of a therapeutically effective amountof aromatic-cationic peptide (e.g., 1-10 mg/kg body weight), such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt. Peptides may beadministered orally, topically, systemically, intravenously,subcutaneously, intraperitoneally, or intramuscularly according tomethods known in the art.

Group 2 receives daily administrations of a therapeutically effectiveamount of a known agent used in the treatment of Friedreich's ataxia,e.g., 100 mg idebenone. The known agent may be administered orally,topically, systemically, intravenously, subcutaneously,intraperitoneally, or intramuscularly according to methods known in theart.

Group 3 receives daily administrations of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂and the same agent as Group 2, wherein the dosage ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and the agent is the same amount used inGroups 1 and 2, respectively. D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and the knownagent may be administered orally, topically, systemically,intravenously, subcutaneously, intraperitoneally, or intramuscularlyaccording to methods known in the art. D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ andthe agent may be administered simultaneously, either as a single pill oras two separate pills, in any order or not simultaneously, e.g.,idebenone is given an hour after treatment withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

The fourth groups receives a similar treatment as Group 3, except athalf the dosage of both D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and the known agent.

Subjects are evaluated weekly for the presence and/or severity of signsand symptoms associated with Friedreich's ataxia, including, but notlimited to, e.g., muscle weakness, especially in the arms and legs, lossof coordination, motor control impairment, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders. Treatments are maintained until such a time as symptoms ofFriedreich's ataxia are ameliorated or eliminated.

Results: It is predicted that Groups 1 and 2 will display reducedseverity of symptoms associated with Friedreich's ataxia. It ispredicted that Group 3 will show a greater reduction in the severity ofsymptoms or elimination of symptoms associated with Friedreich's ataxia.It is predicted that Group 4 will displayed reduced severity of symptomsassociated with Friedreich's ataxia equal to or great than the reductionof symptoms in Groups 1 and 2.

These results will show that the combination of aromatic-cationicpeptides, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, andknown agents used to treat Friedreich's ataxia are useful in thetreatment of Friedreich's ataxia. The synergistic effect of thecombination of the two treatments can lead to a reduced dosage of bothcompounds, thereby reducing possible side effects of the compounds.Accordingly, the peptides are useful in methods comprising administeringaromatic-cationic peptides to a subject in need thereof for thetreatment of Friedreich's ataxia.

Example 5: Treatment of Friedreich's Ataxia UsingD-Arg-2′,6′-Dmt-Lys-Phe-NH₂

This example will demonstrate the use of aromatic-cationic peptides,such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptablesalt thereof, such as acetate or trifluoroacetate salt, in the treatmentof Friedreich's ataxia.

Methods: 24 subjects diagnosed with Friedreich's ataxia are randomlysplit into four groups (3 test groups and 1 control group) with sixsubjects per group. Group 1 receives daily intravenous administrationsof D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ at 0.1 mg/kg of body weight. Group 2receives daily intravenous administrations ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ at 0.5 mg/kg of body weight. Group 3receives daily intravenous administrations ofD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ at 1.0 mg/kg of body weight. Group 4receives daily intravenous administrations of a control peptide at 1.0mg/kg of body weight.

Subjects are selected based on: 1) having a molecular genetic diagnosisof Friedreich's ataxia (FRDA) consisting of a GAA-repeat expansion onboth alleles of the FXN gene; 2) FRDA patients over the age of 18 years;3) subjects must be well enough and willing to provide written informedconsent; and 4) a female subject is eligible to participate if she isof: a) non-childbearing potential defined as pre-menopausal females witha documented tubal ligation or hysterectomy; or postmenopausal definedas 12 months of spontaneous amenorrhea (in questionable cases a bloodsample with simultaneous follicle stimulating hormone (FSH) >40 MlU/mland estradiol <40 pg/ml (<140 pmol/L) is confirmatory); b) child-bearingpotential and agrees to use one of the following contraception methods:abstinence, contraceptive methods with a failure rate of <1%, oralcontraceptive (either combined or progestogen alone), injectableprogestogen, implants of levonorgestrel, estrogenic vaginal ring,percutaneous contraceptive patches, intrauterine device (IUD) orintrauterine system (IUS) that meets the <1% failure rate as stated inthe product label, male partner(s) sterilization (vasectomy withdocumentation of azoospermia) prior to the female subject's entry intothe study, double barrier method, e.g., condom and occlusive cap(diaphragm or cervical/vault caps) plus vaginal spermicidal agent(foam/gel/film/cream/suppository).

FRDA subjects are excluded based on: 1) subjects with significantclinical dysphagia; 2) subjects taking sodium valproate or any otherknown histone deacetylase inhibitor; 3) subject's participating inanother clinical trial or who have done so within 30 days beforescreening; 4) subjects known to be positive for human immunodeficiencyvirus (HIV); 5) subjects with any additional medical condition orillness that, in the opinion of the investigator would interfere withstudy compliance and/or impair the patient's ability to participate orcomplete the study; 6) concurrent diseases or conditions that mayinterfere with study participation or safety include liver disease,bleeding disorders, arrhythmias, organ transplant, organ failure,current neoplasm, poorly controlled diabetes mellitus, poorly controlledhypertension, clinically significant haematological or biochemicalabnormality; 7) subjects with a history of substance abuse (e.g.,alcohol or drug abuse) within the previous 6 months before enrollment;8) subjects with a history of severe allergies; 9) inability to provideinformed consent; 10) female subjects who are lactating or pregnant(positive pre-randomisation serum pregnancy test) or plan to becomepregnant during the study; and 11) subjects unable or unwilling toprovide written informed consent

Subjects are evaluated every two weeks for the presence and/or severityof signs and symptoms associated with Friedreich's ataxia, whichincluding, but are not limited to, e.g., muscle weakness, loss ofcoordination, motor control impairment, vision impairment, hearingimpairment, slurred speech, curvature of the spine, diabetes, and heartdisorders. Treatments and evaluations are maintained for 12 months.

Methods for measuring loss of coordination include, but are not limitedto, Functional Reach Test, Pediatric Clinical Test of SensoryInteraction for Balance, the Pediatric Balance Scale, the Timed “Up &Go” Test, the Timed “Up and Down Stairs” Test, and the measurement ofstatic standing.

Methods for measuring loss of coordination include, but are not limitedto, force control measurements of various muscle groups usingdynamometer in the isometric testing mode.

Results: It is anticipated that Groups 1, 2, and 3 will display reducedseverity of symptoms associated with Friedreich's ataxia as compared toGroup 4. It is also anticipated that Groups 1, 2, and 3 will show a dosedependent reduction in the severity of symptoms associated withFriedreich's ataxia.

These results will show that the combination of aromatic-cationicpeptides, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, andknown agents used to treat Friedreich's ataxia are useful in thetreatment of Friedreich's ataxia. The synergistic effect of thecombination of the two treatments can lead to a reduced dosage of bothcompounds, thereby reducing possible side effects of the compounds.Accordingly, the peptides are useful in methods comprising administeringaromatic-cationic peptides to a subject in need thereof for thetreatment of Friedreich's ataxia.

Example 6: Aromatic-Cationic Peptides Restore Mitochondrial MembranePotential and Translocation of Frataxin into Mitochondria

This example will demonstrate that aromatic-cationic peptides, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, restore mitochondrialmembrane potential and increase translocation of frataxin into themitochondria.

Methods and Materials

Cell line: P131 is a lymphoblast cell line with deficient frataxinexpression. P131 is transfected with a pcDFRDAHA1 vector that containsthe 210 amino acid human frataxin tagged with a HA1 epitope. Thetranscriptional unit is under the control of the CMV immediate-earlypromoter. The plasmid also encodes the geneticin resistance gene forselection of transfectants. The inserted sequence is confirmed by DNAsequencing. Plasmid DNA is prepared using a DNA miniprep commercial kit(Promega, Madison, Wis.). DNA quality is determined by restrictionendonuclease digestion and quantified by UV spectrophotometry.

Transfected lymphoblast line P131 is prepared by growing P131 in freshmedium for 16 hours, and then transiently transfecting P131 with 2 μg/mlpcDFRDAHA1 expression vector or pcDFRDAHA1 empty vector, or 1 μg/ml ofthe reporter gene plasmid pCMV.sport-βgal using DMRIE-C (Life-Tech, CA)according to the manufacturer's protocol for suspension cells. Eachtransfection is performed in triplicate in 6-well plates with 2 μg ofplasmid DNA, 6 μl of DMRIE-C and 2×10⁶ cells mixed in 1.2 ml/well ofOPTI-MEM low-serum medium. Five hours after transfection, fresh culturemedium is added.

24 hours after transfection, cells are stained with X-gal to determinetransfection efficiency and selected with 400 μg/ml geneticin for 12days. Frataxin gene expression is examined by semiquantitative andquantitative RT-PCR and anchored-RT-PCR, western blot and dot blot asdescribed below. Cell lines expressing low (i.e., having similarfrataxin expression levels as cells from a subject diagnosed withFriedreich's ataxia), and high frataxin levels are selected for assays,and aliquots of cells are frozen for experiments. Frataxin mRNAexpression levels are periodically examined by quantitative RT-PCR onthe lightcycler.

Measuring Mitochondrial Potential: Transfected P131 cells are plated ona dish and treated with 0.1 mM t-butyl hydroperoxide (t-BHP), alone orwith 1 nM D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, for 6 hours. Cell untreated witht-BHP and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ are used as a control. Cells arethen treated with 10 μm of dichlorofluorescin (ex/em=485/530) for 30minutes at 37° C., 5% CO₂. The cells are subjected to a wash with HBSSthree time and stained with 20 nM of Mitotracker TMRM (ex/em=550/575 nm)for 15 minutes at 37° C. The cells are then examined by confocal laserscanning microscopy.

Measuring Translocation of Frataxin: Transfected P131 cells that exhibitlow or high expression of frataxin are plated onto six dishes, whereinthree dishes are treated with 1 nM D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and threedishes are not treated, i.e., control cells. After 6 hours, each dish iswashed with wash buffer and fix for staining. Frataxin is fluorescentlytagged by treating the cells with FITC anti-HA1 antibodies for about onehour at room temperature. Each plate is then examined by fluorescencemicroscope (Axiovert™) Transfected P131 cells that exhibit lowexpression of frataxin mimic the disease state of Friedreich's ataxia. Aparallel assay using transfected P131 cells that exhibit high expressionof frataxin is performed.

Results

It is anticipated that t-BHP treated transfected P131 cells withoutD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ treatment will show a loss of TMRMfluorescence, which indicates mitochondrial depolarization. It isanticipated that t-BHP treated transfected P131 cells withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ treatment will show TMRM fluorescence, whichindicates prevention of mitochondrial depolarization and restoration ofthe membrane potential. It is anticipated that cell not treated witheither t-BHP or D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ will also show a loss ofTMRM fluorescence, however the loss will be less than the t-BHP onlytreated cells.

It is anticipated that transfected P131 cells that exhibit low and highfrataxin expression level when treated with D-Arg-2′,6′-Dmt-Lys-Phe-NH₂will show an increase in frataxin localized to the inner membrane of themitochondria as compared to transfected P131 cells not treated withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

These results will show that D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ is useful forrestoring mitochondrial membrane potential. The results will also showthat maintaining the mitochondrial membrane potential results in thetranslocation of frataxin to the inner mitochondrial membrane.

Example 7: Use of Aromatic-Cationic Peptides in Treating MitochondrialIron Loading in Friedreich's Ataxia Mouse Model

This example will demonstrate the use of aromatic-cationic peptides,such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptablesalt thereof, such as acetate or trifluoroacetate salt, in treatingmitochondrial iron loading in Friedreich's ataxia.

Mouse model. This example uses the muscle creatine kinase (MCK)conditional frataxin knockout mice described by Puccio et al., Nat.Genet. 27:181-186 (2001). In this model, the tissue-specific Cretransgene under the control of MCK promoter results in the conditionaldeletion of frataxin in only the heart and skeletal muscle.

Eight-week-old mutant mice are administered a daily dose of 0.25mg/kg/day of D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or saline vehicle only(control) subcutaneously for two weeks. Total RNA is isolated fromhearts of two 10-week-old wild-type mice, two 10-week-old untreatedmutant mice and two 10-week-old treated mutant mice. Total RNA isisolated using TRIzol (Invitrogen). First-strand cDNA synthesis andbiotin-labeled cRNA are performed and hybridized to the mouse AffymetrixGeneChip 430 2.0. A 2-phase strategy is used to identify differentiallyexpressed genes. First, genome-wide screening is performed usingAffymetrix GeneChips. Then, low-level analysis is performed withAffymetrix GeneChip Operating Software 1.3.0, followed by the GC robustmultiarray average (GCRMA) method for background correction andquantile-quantile normalization of expression. Tukey's method formultiple pairwise comparisons is applied to acquire fold-changeestimations. Tests for significance are calculated and adjusted formultiple comparisons by controlling the false discovery rate at 5%.

Definitive evidence of differential expression is obtained from RT-PCRassessment of samples used for the microarray analysis and at least 3other independent samples. Principal component analysis is performed bystandard methods. Western blot analysis is performed using antibodiesagainst frataxin (US Biological); Tfr1 (Invitrogen); Fpn1 (D. Haile,University of Texas Health Science Center); Hmox1 (AssayDesigns); Sdha,Gapdh, and Iscu1/2 (Santa Cruz Biotechnology); Fech (H. Dailey,University of Georgia, Biomedical and Health Sciences Institute); Hfe2(S. Parkkila, University of Tampere, Institute of Medical Technology);Nfs1, Uros, and Alad (Abnova); Sec15l1 (N. C. Andrews, Duke University);F11, Fth1, Ftmt (S. Levi, San Raffaele Institute); and Hif1α (BDBiosciences).

For heme assays, hearts are exhaustively perfused and washed with PBS(0.2% heparin at 37° C.) to remove blood. After homogenization, heme isquantified using the QuantiChrom Heme Assay (BioAssay Systems). Tissueiron is measured via inductively coupled plasma atomic emissionspectrometry

For iron loading measurement assays, hearts are exhaustively perfusedand washed with PBS (0.2% heparin at 37° C.) to remove blood.Mitochondria from the hearts are isolated using a mitochondrialisolation kit (Thermo Scientific, Rockford, Ill.). The ironconcentration of the mitochondria is determined by the Ferene S-basedIron Assay Kit (BioVision, Milpitas, Calif.) according to themanufacturer's protocol.

It is anticipated that untreated mutant mice will exhibit decreasedexpression of genes involved in heme synthesis, iron-sulfur clusterassembly, and iron storage (FRDA Control) as compared to wild-type mice(Normal). However, it is anticipated that mutant mice treated withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ will show expression levels that are similarto normal subjects with respect to genes involved in these threemitochondrial iron utilization pathways. It is further expected thatadministration of the present technology will have synergistic effectsin this regard. It is also anticipated that mice treated withD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ will show an decrease in iron within theisolated mitochondria as compared to untreated mice.

These results will show that aromatic-cationic peptides of the presenttechnology are useful in treating mitochondrial iron loading inFriedreich's ataxia or in subjects with lower frataxin expression oractivity.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for treating heart disease in a subjecthaving Friedreich's ataxia, comprising administering to the subject atherapeutically effective amount of the peptideD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the peptide is administereddaily for 6 weeks or more.
 3. The method of claim 1, wherein the peptideis administered daily for 12 weeks or more.
 4. The method of claim 1,wherein the heart disease comprises a condition selected from the groupconsisting of hypertrophic cardiomyopathy, myocardial fibrosis, andcardiac failure.
 5. The method of claim 1, wherein the heart diseasecomprises hypertrophic cardiomyopathy.
 6. The method of claim 1, whereinthe heart disease comprises myocardial fibrosis.
 7. The method of claim1, wherein the heart disease comprises cardiac failure.
 8. The method ofclaim 1, wherein the heart disease comprises a heart rhythm abnormalitycondition selected from the group consisting of tachycardia and heartblock.
 9. The method of claim 1, wherein the subject suffers symptomsselected from the group consisting of chest pain, shortness of breath,and heart palpitations.
 10. The method of claim 1, wherein the subjectis human.
 11. The method of claim 1, wherein the peptide is administeredorally, topically, systemically, intravenously, subcutaneously,intraperitoneally, or intramuscularly.
 12. The method of claim 1,further comprising separately, sequentially or simultaneouslyadministering to the subject one or more additional therapeutic agentsselected from the group consisting of ACE inhibitors, digoxin,enalapril, or lisinopril, diuretics, beta blockers, idebenone,deferiprone, and insulin.
 13. The method of claim 1, wherein thepharmaceutically acceptable salt comprises acetate or trifluoroacetatesalt.
 14. The method of claim 12, wherein administering the peptide andthe additional therapeutic agent has a synergistic effect in thetreatment of Friedreich's ataxia.